Dopamine-Dependent Neurodegeneration in Rats Induced by Viral Vector-Mediated Overexpression of the Parkin Target Protein, Cdcrel-1

Dopamine-dependent neurodegeneration in rats induced by viral vector-mediated overexpression of the parkin target protein, CDCrel-1

Zhizhong Dong*, Boris Ferger†, Jean-Charles Paterna*, Denise Vogel*, Sven Furler*, Maribel Osinde*, Joram Feldon†, and Hansruedi Bu¨ eler*‡

*Institute of Molecular Biology, University of Zurich, 8057 Zurich, Switzerland; and †Behavioral Neurobiology Laboratory, Swiss Federal Institute of Technology (ETH), 8603 Schwerzenbach, Switzerland

Edited by L. L. Iversen, University of Oxford, Oxford, United Kingdom, and approved August 18, 2003 (received for review May 19, 2003) Mutations in the parkin gene are linked to autosomal-recessive We have shown earlier that recombinant adeno-associated juvenile parkinsonism (AR-JP). Parkin functions as a ubiquitin viruses (rAAV) carrying the neuron-specific platelet-derived protein ligase in the degradation of several proteins, including the growth factor ␤-chain (PDGF-␤) promoter confer efficient and neuron-speciﬁc septin CDCrel-1. AR-JP-associated parkin mutations long-lasting transgene expression throughout the rat dopamine inhibit ubiquitination and degradation of CDCrel-1 and other system without causing toxicity or inflammation (23, 24). Here, parkin target proteins. Here we show that recombinant adeno- we exploited rAAV-mediated gene transfer to investigate associated virus-mediated CDCrel-1 gene transfer to the substantia whether CDCrel-1 may be detrimental to dopamine neurons. nigra of rats results in a rapid onset (6–10 days) of nigral and For this purpose, we expressed human CDCrel-1 or enhanced striatal CDCrel-1 expression that is followed by a progressive loss GFP (EGFP, control) unilaterally in the nigrostriatal system of of nigral dopaminergic neurons and a decline of the striatal rats by stereotactic nigral delivery of rAAV vectors and studied dopamine levels. In contrast, neurons of the globus pallidus are the effects of the two proteins on the survival of dopamine spared from CDCrel-1 toxicity. Furthermore, CDCrel-1 inhibits the neurons and on the levels of dopamine and its metabolites in the release of dopamine from stably-transfected PC12 cells, and phar- STR. Our results show that CDCrel-1 expression induces selec- macological inhibition of tyrosine hydroxylase and dopamine syn- tive dopamine neurodegeneration that can be blocked by inhi- thesis in rats prevents CDCrel-1-induced nigral neurodegeneration. bition of tyrosine hydroxylase (TH) and dopamine biosynthesis. These results show that CDCrel-1 overexpression exerts dopamine- We also find that CDCrel-1 inhibits dopamine release from dependent neurotoxicity and suggest that inhibition of dopamine PC12 cells, suggesting that a block in dopamine secretion may secretion by CDCrel-1 may contribute to the development of AR-JP. contribute to the development of AR-JP. Materials and Methods arkinson’s disease (PD) is characterized by the progressive Plasmid Constructions. The human CDCrel-1 ORF (GenBank loss of dopaminergic neurons in the substantia nigra pars P accession no. U74628) was amplified by RT-PCR with primers compacta (SNc), declining levels of dopamine in the striatum 5Ј-CGACGCGTGCCACCATGAGCACAGGCCTG-3Ј and 5Ј- (STR), and motor impairments, including bradykinesia, rigidity, CGACGCGTTCACTGGTCCTGCATCTGCTG-3Ј, and in- and tremor (1, 2). Several genetic loci have been implicated in serted into plasmid psubPDGF-WPRE (24) to generate psub- the pathogenesis of familial forms of PD (3, 4). Two missense ␣ PDGF-CDCrel-1-WPRE. psubPDGF-EGFP-WPRE was mutations in the -synuclein gene are linked to a rare, dominant generated by insertion of the EGFP fragment derived from form of PD (5, 6). In addition, a variety of mutations in the parkin pEGFP-N1 (Clontech) into psubPDGF-WPRE. The CDCrel-1 gene are responsible for autosomal-recessive juvenile parkin- coding sequence was fused to the EGFP ORF within pEGFP-N1 sonism (AR-JP) (7, 8), and a dominant mutation in the ubiquitin (Clontech), and the fragment encoding the fusion protein was C-terminal hydrolase gene L1 (UCH-L1) has been linked to PD subsequently transferred to psubPDGF-WPRE. in one pedigree (9). Most recently, mutations in the DJ-1 gene (10), and the NR4A2 gene (11) encoding the nuclear receptor Generation and Purification of rAAV. High-titer, helper virus-free and transcription factor Nurr-1, have been reported to segregate rAAV-2 were generated with plasmid pDG (25) and purified as with familial PD. described (25, 26). The rAAV stocks were sterile-filtered (0.2 ␮m) Parkin functions as a ubiquitin protein ligase to promote the and titrated by slot blot hybridization (56). Titers (genomes per ml) degradation of specific target proteins (12, 13). The first parkin were 7.1 ϫ 1011 (rAAV-PDGF-CDCrel-1), 8.3 ϫ 1011 (rAAV- target to be identified was CDCrel-1 (13), a protein of the septin PDGF-EGFP), and 3.6 ϫ 1011 (rAAV-PDGF-CDCrel-1͞EGFP). gene family that is predominantly expressed in the brain and localized mainly to presynaptic axon terminals of inhibitory Animals, Stereotactic Surgery, and ␣-Methyl-DL-Tyrosine (AMPT) neurons (14–16). CDCrel-1 associates with membranes includ- Treatment. All animals were housed and treated according to ing synaptic vesicles (15) and directly binds to the soluble institutional and national guidelines. Male Wistar rats (285 Ϯ N-ethylmaleimide-sensitive factor attachment protein receptor 22 g) were stereotactically injected with 2 ␮l of rAAV dorsal (SNARE) protein syntaxin-1 (17). Subsequently, several other parkin substrates were identified, including a G protein-coupled receptor termed Pael-R (18), an unusual O-glycosylated 22-kDa This paper was submitted directly (Track II) to the PNAS ofﬁce. form of ␣-synuclein (19), synphilin-1 (20), as well as ␣- and Abbreviations: PD, Parkinson’s disease; AR-JP, autosomal-recessive juvenile parkinsonism; ␤-tubulin (21). AR-JP-linked parkin mutants fail to ubiquitinate PDGF, platelet-derived growth factor; SNc, substantia nigra pars compacta; STR, striatum; rAAV, recombinant adeno-associated viruses; TH, tyrosine hydroxylase; EGFP, enhanced and degrade the parkin targets, suggesting that AR-JP is caused GFP; DOPAC, dihydroxyphenylacetic acid; HVA, homovanillic acid; GP, globus pallidus; by the accumulation of proteins that are ultimately toxic to AMPT, ␣-methyl-DL-tyrosine; SNARE, soluble N-ethylmaleimide-sensitive factor attachment dopamine neurons (13, 19, 20). Interestingly, parkin is able to protein receptor. protect neurons from diverse cellular insults, suggesting a central ‡To whom correspondence should be addressed. E-mail: [email protected]. role for parkin in maintaining dopamine neuron integrity (22). © 2003 by The National Academy of Sciences of the USA

12438–12443 ͉ PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 www.pnas.org͞cgi͞doi͞10.1073͞pnas.2132992100 Downloaded by guest on September 30, 2021 to the right SNc at two coordinates anterio-posterior (AP) Ϫ5.3 mm, medio-lateral (ML) ϩ 1.8 mm, dorso-ventral (DV) Ϫ7.7 mm and AP Ϫ5.3 mm, ML ϩ 2.5 mm, DV Ϫ7.3 mm, relative to bregma). In the globus pallidus (GP), 2 ␮lofvirus solution was injected at each of the following two coordinates: AP Ϫ0.90 mm͞Ϫ1.30 mm, ML ϩ 2.8 mm͞ϩ3.2 mm, DV Ϫ6.3 mm͞Ϫ6.6 mm. To inhibit TH and dopamine synthesis, animals were injected i.p. twice per day with 175 mg͞kg AMPT-HCl (Fluka) dissolved in 0.9% NaCl (saline).

Tissue Preparation and Determination of Dopamine and Dopamine Metabolites. Rats were killed by decapitation at the indicated time points after rAAV injections. Brains were rapidly removed and placed on an ice-cold plate for dissection of the neostriatum. Tissues were processed, and dopamine and its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were analyzed by using reverse-phase ion-pair chroma- tography combined with electrochemical detection under iso- cratic conditions, as described (27).

Immunohistochemistry. Coronal cryosections (20 ␮m thick) containing the SNc were prepared from 4% paraformaldehyde-fixed brains. Primary Abs included CDCrel-1-specific hybridoma supernatant SP18 (1:50) (16), mouse anti-rat TH (1:1,000) (Pharm- ingen), rabbit anti-dopamine transporter (1:100) (Chemicon), rabbit anti-glutamic acid decarboxylase (1:1,000) (Affinity, Exeter, U.K.), and mouse anti-NeuN (1:200) (Chemicon). Fig. 1. AAV-mediated expression of EGFP, CDCrel-1, and CDCrel-1͞EGFP in

Secondary Cy3- (1:500), FITC- (1:400), and biotin-conjugated the nigrostriatal system of rats. Transgene products were detected by native NEUROSCIENCE (1:1,000) immunoglobulins were from Milan Analytica, La (EGFP) fluorescence (A and G) or immunohistochemistry with an Ab against CDCrel-1 (D). Dopamine neurons in the same sections were identified with a Roche, Switzerland. Biotin-conjugated Abs were used in com- mAb against rat TH (B, E, and H). Dopamine neurons expressing EGFP (C), bination with diaminobenzidine staining (ABC vector kit, Vec- ͞ ͞ CDCrel-1 (F), or CDCrel-1 EGFP (I) are shown. (J) A higher magnification of tor Laboratories). Expression of EGFP and the CDCrel-1 CDCrel-1-transduced dopamine neurons. Expression is shown at 10 days (D–J) EGFP fusion protein in the substantia nigra were detected by or 42 days (A–C) after rAAV injection. No transgene expression is found in native fluorescence microscopy (without Abs). dopamine neuron bodies of the uninjected SNc (data not shown), but endogenous CDCrel-1 is strongly expressed in fibers of the SNr in both uninjected Western Blot Analysis. Virus-injected striatal hemispheres were (data not shown) and injected (D and J) hemispheres. Striatal expression of dissected out and homogenized in 0.3 ml of 4% Triton X-100, 150 rAAV-derived CDCrel-1͞EGFP at 11 and 14 days (n ϭ 4 rats͞time) after trans- mM NaCl, and 10 mM Tris⅐HCl (pH 8.0) containing protease duction of the ipsilateral SNc (K). No transgene expression was found in the uninjected striatal hemispheres of the same animals (data not shown). (Bar ϭ inhibitor mixture (Sigma), and the homogenates were clarified 0.2 mm in A–I and 0.1 mm in J.) by centrifugation for 15 min at 1,000 ϫ g. Total protein (75 ␮g) was separated by SDS͞PAGE and analyzed by Western blot with the SP18 mAb against CDCrel-1 (1:400) to detect endogenous CDCrel-1. Stably-transfected PC12 cell populations (pools of CDCrel-1, or with 1:500 diluted anti-GFP mAb (Sigma, clone ͞ ͞ G418-resistant colonies) were selected with 0.3 mg ml G418. GFP-20) to detect rAAV-derived CDCrel-1 EGFP, followed by CDCrel-1 expression was detected with the SP18 Ab as described incubation with 1:15,000 diluted horseradish peroxidase- above. EGFP expression was detected by fluorescence micros- conjugated goat anti-mouse IgG (Roche Diagnostics). Bands copy in the FITC channel. were visualized by enhanced chemiluminescence (Pierce). Determination of Intracellular and Secreted Dopamine in Stably Quantification of Neurons and TH Fiber Density. Diaminobenzidine- Transfected PC12 Cells. KCl-evoked dopamine release was mea- stained, TH-positive neurons in SNc were counted in 20-␮m- sured as described (28, 29). To measure intracellular dopamine, thick coronal sections containing the medial terminal nucleus of the cells were homogenized by sonication in cold 0.3 M HClO the accessory optical tract to distinguish between dopamine 4 neurons of the SNc and the ventral tegmental area. Counting was and incubated on ice for 15 min, and the homogenates were centrifuged at 16,000 ϫ g for 15 min at 4°C. Supernatants were carried out at ϫ40 magnification by using NEUROLUCIDA software (MicroBrightField, Colchester, VT). Quantification of used for HPLC analysis of dopamine content. Pellets (precipi- tated proteins) were washed once in PBS and solubilized in 0.1 NeuN-positive neurons in the GP was carried out by the same ⅐ method in coronal sections containing a visible needle tract ml 1% Triton X-100, 150 mM NaCl, and 10 mM Tris HCl (pH (from the virus injection). Optical densities of the TH- 8.0) containing protease inhibitor mixture (Sigma) for protein immunoreactive fibers in the STR were measured by using determination (BCA kit, Pierce). IMAGE software (National Institutes of Health). For each animal, Results the optical density was measured in four different striatal sections. Optical density readings were corrected by subtraction AAV-Mediated Expression of EGFP and CDCrel-1 in the Nigrostriatal of nonspecific background density, determined in adjacent sec- System of Rats. High-titer rAAV expressing CDCrel-1, CDCrel- tions that were incubated without the primary TH Ab. 1͞EGFP fusion protein, or EGFP (control) from the neuron- specific PDGF-␤ promoter were injected stereotactically dorsal Generation and Characterization of Stably Transfected PC12 Popula- to the SNc of the right hemisphere of rats. Expression of all tions. PC12 cells were electroporated (280 V, 960 ␮F) with transgenes could be detected in TH-positive neurons as early as pcDNA3 expression vectors (Invitrogen) encoding EGFP or 6 days after rAAV injection. At 10 days, the majority of

Dong et al. PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 ͉ 12439 Downloaded by guest on September 30, 2021 Fig. 2. CDCrel-1 induces a progressive striatal depletion of dopamine and its metabolites. Concentrations (mean Ϯ SEM) of dopamine (A), DOPAC (B), and HVA (C) in injected (black bars) and uninjected (dotted bars) striatal hemispheres were determined between 6 and 42 days after supranigral injection of Fig. 3. TH-positive neurons in the substantia nigra of rats. Dopamine the rAAV-CDCrel-1 vector. Rats injected with the rAAV-EGFP virus were ana- neurons in normal (A and C) and virus-injected (B and D) hemispheres of the lyzed at 42 days. (D) Dopamine turnover. CDCrel-1: n ϭ 4 rats at 6, 10, 15, 20, SNc 42 days after injection of rAAV-EGFP (A and B) and rAAV-CDCrel-1 (C and and 26 days, and n ϭ 11 rats at 42 days. EGFP: n ϭ 12 rats. Data were evaluated D). (E) Survival of TH-positive neurons in rAAV-injected hemispheres, relative statistically by ANOVA and Fisher’s probable least-squares difference test (***, to uninjected control side (see Materials and Methods). Data are expressed as Ϯ ϭ P Ͻ 0.001; **, P Ͻ 0.01; *, P Ͻ 0.05, compared to the uninjected hemisphere). mean SD of three to four sections per rat. rAAV-EGFP: n 3 rats. rAAV- CDCrel-1: n ϭ 4 rats.

dopamine neurons expressed CDCrel-1 (Fig. 1 D–F) and CD- Crel-1͞EGFP (Fig. 1 G–I). The presence of CDCrel-1 in cell injection of the rAAV-CDCrel-1 virus, the striatal levels of bodies suggests that the protein was overexpressed because dopamine were reduced by 53.5% in the injected hemisphere CDCrel-1 is not normally detected in nigral dopamine neurons relative to the untreated brain side. Likewise, the levels of but it is present at low levels in fibers of the STR (14). At 10 days, DOPAC and HVA were reduced by 32.9% and 38.2%, and the transgene-expressing neurons appeared healthy and had dopamine turnover was increased by a factor of 1.4 (40% normal morphology (Fig. 1J). At 15 days, and more evidently at increase). The levels of the various measured catecholamines 20 and 26 days, the number of CDCrel-1-expressing neurons and were further reduced at 20 days after rAAV delivery (Fig. 2 TH-positive neurons were substantially reduced in the injected A–C), but the relative reductions of the individual cat- hemisphere (data not shown). These results show that the rAAV echolamines remained similar until day 42. At this time point, vectors confer early transgene expression and suggest that a the levels of dopamine, DOPAC, and HVA were reduced by progressive loss of dopamine neurons expressing CDCrel-1 or 73.9%, 59.2%, and 47.8%, respectively, in the rAAV-CDCrel-1 the CDCrel-1͞EGFP fusion protein began between 10 and 20 treated hemispheres, and the dopamine turnover was in- days after rAAV delivery. creased by a factor of 2.2 (Fig. 2). Injection of a 5-fold diluted Because CDCrel-1 is a synaptic vesicle-associated protein, we rAAV-CDCrel-1 virus stock was less toxic but still caused carried out Western blots to determine whether nigral injection significant reductions in striatal catecholamines (dopamine: of the rAAV vectors resulted in protein expression in the STR, Ϫ46.4%; DOPAC: Ϫ34.9%; HVA: Ϫ31.0%). the target area of dopamine neurons. Because CDCrel-1 is Quantitation of dopamine neurons at 42 days revealed an expressed in the STR (14, 15), we analyzed the striatal expression average loss of 78.5 Ϯ 6.9% of the TH-positive neurons in the of CDCrel-1͞EGFP by using an Ab against EGFP to specifically virus-injected hemisphere (Fig. 3 D and E), which correlated well detect rAAV-derived protein. The same samples were also with the decline of the dopamine levels (73.9%, Fig. 2A). analyzed with the SP18 Ab to reveal endogenous CDCrel-1 Immunohistochemistry with an Ab against the dopamine trans- expression (internal standard). Striatal CDCrel-1͞EGFP was porter, and the pan-neuronal nuclear marker NeuN yielded detected at both 11 and 14 days after virus injection (Fig. 1K), similar results, indicating that actual neuron loss and not simply with higher levels at 14 days, consistent with the time-dependent down-regulation of TH expression had occurred (data not increase in rAAV-mediated gene expression. Thus, CDCrel-1 is shown). In contrast, EGFP had no adverse effect on either expressed in striatal axons before the onset of nigral dopamine dopamine neuron survival (Fig. 3 B and E) or the amounts of the neuron loss. analyzed catecholamines in the STR (Fig. 2), despite efficient and sustained EGFP expression in the majority of dopamine CDCrel-1 Induces Dopaminergic Neurodegeneration and a Reduction neurons (Fig. 1 A–C). These results, taken together with the of Dopamine and Its Metabolites in the STR. To study the conse- expression data presented above (Fig. 1), show that an early quences of CDCrel-1 expression on the rat nigrostriatal sys- but transient phase of CDCrel-1 expression in dopamine neurons tem, a time course analysis (days 6–42) of the levels of striatal is followed by a dramatic decline of striatal dopamine levels dopamine and its metabolites in virus-injected and uninjected that is accompanied by the loss of dopamine neurons in the hemispheres was carried out (Fig. 2). Significantly reduced injected SNc. levels of dopamine (Fig. 2A), DOPAC (Fig. 2B), and HVA (Fig. 2C) were observed between days 15 and 42 after rAAV CDCrel-1 Inhibits Dopamine Release from PC12 Cells. CDCrel-1 binds delivery. Moreover, dopamine turnover was significantly in- to syntaxin-1 and inhibits the secretion of (transfected) growth creased at all these time points (Fig. 2D). Fifteen days after hormone in the insulinoma cell line HIT-T15 (17). This has led

12440 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.2132992100 Dong et al. Downloaded by guest on September 30, 2021 Fig. 4. CDCrel-1 inhibits dopamine secretion. Shown is expression of CDCrel-1 (A) and EGFP (B) in stably transfected PC12 cell populations (pools of G418-resistant colonies). (C) Secreted dopamine was measured after incubation of the cells in medium containing 80 mM KCl (n ϭ 12 wells for each population) as described (28, 31). (D) Intracellular dopamine (n ϭ 8 wells for Fig. 5. Inhibition of dopamine synthesis prevents CDCrel-1-induced neuro- each populations). Data are normalized to protein content and expressed as degeneration. Rats were treated with the TH inhibitor AMPT or saline (as a mean Ϯ SD. ***, P Ͻ 0.001. The experiments were repeated several times with control) beginning 9 days after rAAV-CDCrel-1 injection. To conﬁrm the similar results. potency of AMPT, dopamine levels were compared by HPLC in the uninjected

hemispheres of saline- and AMPT-treated rats at 12 days after rAAV injection NEUROSCIENCE (n ϭ 4 rats per group) (A). Shown are TH-positive neurons (B) and percentage to the proposal that an increased abundance of CDCrel-1 due to of TH ﬁber density (C) at day 15 in rAAV-CDCrel-1-injected and -uninjected the impairment of parkin may inhibit dopamine release and brain hemispheres of saline- and AMPT-treated rats (neuron counts: n ϭ 4 rats contribute to a Parkinsonian state (13, 30). However, this per group, four to six sections per rat; TH ﬁber density: n ϭ 4 rats per group, hypothesis has not been addressed experimentally in cells that four sections per rat). (D) Expression of CDCrel-1 in nigral dopamine neurons naturally produce and secrete dopamine. Therefore, we studied of AMPT-treated rats at day 15. Data were evaluated statistically by ANOVA and Fisher’s probable least-squares difference test (***, P Ͻ 0.001; **, P Ͻ 0.01; the effect of CDCrel-1 overexpression on the secretion of , P Ͻ 0.05, compared to the uninjected hemisphere). dopamine by rat PC12 neuroendocrine cells. These cells synthe- * size and store dopamine in secretory vesicles and can be depo- larized to release dopamine into the medium (28, 31). We were continued up to day 14. At day 15, all rats were killed to generated PC12 cell populations stably expressing CDCrel-1 or determine nigral dopamine neuron survival and striatal TH fiber EGFP and confirmed protein expression by Western blot and density. Both neuron survival and TH fiber density were signif- fluorescence microscopy (Fig. 4 A and B). Potassium chloride icantly reduced in the rAAV-CDCrel-1 injected hemisphere of (KCl)-evoked secretion of dopamine was assessed by quantitat- saline-treated rats (Fig. 5 B and C). In contrast, in AMPT- ing the dopamine levels in the extracellular medium of PC12 cells treated rats TH neuron numbers and TH fiber density were by HPLC (28, 31). The extracellular medium of PC12 cells comparable in virus-injected and uninjected hemispheres (Fig. 5 expressing CDCrel-1 contained significantly less dopamine than B and C). To rule out the possibility that AMPT interfered medium from control PC12 cells expressing EGFP (Fig. 4C). with the expression of CDCrel-1, nigral sections from the same Quantitation of intracellular dopamine levels revealed that both animals were analyzed for expression of CDCrel-1. CDCrel-1 CDCrel-1 and EGFP-expressing PC12 cells contained compa- was indeed expressed in nigral dopamine neurons at day 15 of rable amounts of dopamine (Fig. 4D). Therefore, the reduced AMPT-treated rats (Fig. 5D). Furthermore, AMPT treatment extracellular dopamine levels reflect a decrease in dopamine secretion by PC12 cells expressing CDCrel-1. did not result in up-regulation of TH expression, because the preservation of neuron survival was also observed by NeuN Inhibition of TH Prevents CDCrel-1-Induced Neurodegeneration. In staining (data not shown). Collectively, these results show that vivo, inhibition of dopamine secretion may ultimately cause CDCrel-1 toxicity depends on dopamine. neuronal loss via dopamine-dependent toxicity (32). To address whether CDCrel-1 induced neurodegeneration depends on do- Nondopaminergic Neurons Are Unaffected by CDCrel-1 Overexpres- pamine, we pharmacologically inhibited dopamine synthesis in sion. To further test the requirement of dopamine for CDCrel-1 rats with AMPT, a highly specific inhibitor of TH. Initial toxicity, we unilaterally injected an other brain area, namely the ͞ experiments showed that repeated injections of AMPT stably GP, with the rAAV-EGFP, rAAV-CDCrel-1 EGFP, and ϭ ͞ reduced striatal dopamine levels to Ϸ30% of normal amounts rAAV-CDCrel-1 vectors (n 3 rats per vector type). CDCrel-1 over the course of 4 treatment days (data not shown). Based on EGFP (Fig. 6A) and EGFP (not shown) were readily detected in these findings, 16 rats were injected unilaterally with the rAAV- cells of the injected GP. Colocalization with the neuronal nuclear CDCrel-1 virus above the SNc. Beginning at day 9 after virus marker NeuN (Fig. 6B) revealed that GP neurons were effi- injection, eight rats were treated with AMPT and eight rats were ciently transduced (Fig. 6C). However, in contrast to the SNc, injected with saline as a control. At day 12, four rats of each CDCrel-1 did not cause a loss of neurons in the GP, as revealed group were killed for HPLC analysis of striatal dopamine to by immunohistochemistry for NeuN-positive cells (Fig. 6 D–G). confirm that AMPT reduced dopamine synthesis (Fig. 5A). For This result was further confirmed by the quantitation of NeuN- the remaining four rats of each group, AMPT or saline injections positive neurons in injected and uninjected GP of three rats each

Dong et al. PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 ͉ 12441 Downloaded by guest on September 30, 2021 metabolites in the STR. Importantly, CDCrel-1 toxicity depends on dopamine because pharmacological reduction of dopamine synthesis with a specific inhibitor of TH prevented CDCrel-1 induced dopamine neurodegeneration, and non- dopamine neurons of the GP were spared from CDCrel-1 toxicity. CDCrel-1 is a member of the phylogenetically conserved septin gene family (36–39) and is expressed predominantly in the brain (15, 40). In mammals, at least 10 septins have been identified to date. Several of them are associated with the sec6͞8 (exocyst) complex purified from rat brain (41), suggesting a role for these proteins in the recruitment or docking of vesicles to specific sites on the plasma membrane. Indeed, it has recently been shown that septin organization is con- trolled by Cdc42 effector proteins termed Borgs, linking the septins with proteins that regulate cell polarity, cytoskeletal remodeling, and vesicle transport (42). How might the toxicity of CDCrel-1 for dopamine neurons come about? For neurotransmitter release, synaptic vesicles must dock to and fuse with the presynaptic membrane. These processes depend on the formation of the SNARE complex, which consists of the presynaptic membrane proteins syntaxin-1 and SNAP-25 and the synaptic vesicle proteins synaptobrevin and synaptotagmin. Because CDCrel-1 can directly bind to syntaxin-1, it may Fig. 6. Neurons in the GP are unaffected by CDCrel-1 expression. Expression interfere with the formation of a functional SNARE complex of CDCrel-1͞EGFP (A) in NeuN-positive neurons (B) of the rAAV-injected GP 20 and thus inhibit neurotransmitter release. This hypothesis is days after virus delivery is shown. (C) Overlay. (D–G) Normal (D and F) and consistent with the fact that botulinum toxin C1 (which cleaves rAAV-CDCrel-1-injected (E and G, needle tract indicated in E) GP, showing that syntaxin-1) and another syntaxin-1-interacting protein, the overall number of NeuN-positive neurons is comparable between normal and injected hemispheres. (H) Quantitation of NeuN-positive neurons Munc-18 (43), inhibit exocytosis. Indeed, CDCrel-1 has been (mean Ϯ SD) in normal and virus-injected GP. No difference in the neuronal shown to reduce secretion in a pancreatic cell line (17), null numbers was observed between injected and normal sides (n ϭ 3 rats per and exocytosis in platelets from CDCrel-1 mice can be group; three sections per rat). triggered by subthreshold levels of collagen (44), suggesting that CDCrel-1 exerts an inhibitory effect on exocytosis. How- ever, the relevance of these findings with respect to the role of injected with either the rAAV-CDCrel-1 or rAAV-EGFP con- CDCrel-1 for the development of PD has been unclear. PC12 trol vector (Fig. 6H). cells have been widely used as a catecholaminergic model In addition, in the injected SNc, the loss of TH immunore- system, because they synthesize and store dopamine in secre- activity was paralleled by a loss of transgene-expressing neuronal tory vesicles, and neurotransmitter release can be evoked by cell bodies in rAAV-CDCrel-1- and rAAV-CDCrel-1͞EGFP- depolarization (28, 31). Our finding that CDCrel-1 decreases injected rats, whereas neurons located dorsally (mesencephalic dopamine exocytosis from PC12 cells indicates that CDCrel-1 tegmentum) and ventrally (substantia nigra pars reticulata, SNr) may be a negative regulator of dopamine neurotransmission to the SNc continued to express the transgene products in the and provides a mechanism by which CDCrel-1 may contribute same animals. Finally, although glutamic acid decarboxylase- to the development of PD. Chronic inhibition of dopamine positive neurons were also transduced with CDCrel-1, their release may ultimately cause dopamine-mediated neuron number was comparable in rAAV-injected and uninjected hemi- death (32). An important role of dopamine for CDCrel-1 spheres (but on both sides the number of glutamic acid decar- toxicity is supported by our results that inhibition of dopamine boxylase-positive neuronal bodies was low because the Ab synthesis by AMPT prevented dopamine neuron loss, and that detects glutamic acid decarboxylase mainly in fibers) (data not GP neurons (lacking dopamine) tolerated CDCrel-1 expres- shown). Taken together, these results indicate that CDCrel-1 sion in the absence of toxicity. Damage by dopamine may be ␣ selectively kills dopamine neurons of the SNc and support an further enhanced by endogenously expressed (rat) -synuclein, important role of dopamine for CDCrel-1 toxicity. as it has recently been shown that dopamine toxicity is potentiated by ␣-synuclein in primary midbrain dopamine Discussion neurons (45). Finally, reduced dopamine secretion may result ␣ There is growing evidence that failure of the ubiquitin- in increased oxidative ligation of dopamine to -synuclein and thus in the stabilization of protofibrillar (nonaggregated) proteasome system is critically involved in the development of ␣ both familial and sporadic PD (33–35). Several of the AR-JP- -synuclein intermediates (46), which have been suggested to be neurotoxic by their ability to permeabilize synaptic vesicles associated mutations have been shown to abolish the ubiquitin § protein ligase activity of parkin and thus the degradation of (46–49). parkin target proteins. These data suggest that the accumu- Although PC12 cells expressing CDCrel-1 survived, a rapid lation of certain parkin substrates may ultimately cause do- dopamine neuron loss was observed in rAAV-CDCrel-1- pamine neuron death and the development of AR-JP. To test injected rats. We believe that this difference is due to the efficient tropism of AAV vectors for the transduction of dopa- this hypothesis, we have investigated the consequences of mine neurons in vivo, which leads to high-level protein expres- virus-mediated expression of human CDCrel-1, the first parkin sion throughout the nigrostriatal system of rats (23, 24). This target identified, on the survival of dopamine neurons in rats. We find that rAAV-mediated expression of CDCrel-1 in the rat SNc induces a massive degeneration of the dopamine §Lashuel, H. A., Hartley, D., Petre, B. M., Walz, T. & Lansbury, P. T., Jr. (2002) Nature 418, 291 neurons and a corresponding decline of dopamine and its (abstr.).

12442 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.2132992100 Dong et al. Downloaded by guest on September 30, 2021 notion is supported by a recent study demonstrating that rAAV- responsible for reduced dopamine secretion and to substantiate mediated targeted overexpression of ␣-synuclein also causes a causative pathological role of CDCrel-1 in humans affected dopamine neuron loss (50), whereas several lines of ␣-synuclein with AR-JP. transgenic mice failed to develop nigral TH neuron pathology (51–55). We thank Andreas Leng for assistance with HPLC analysis and In summary, our findings show that CDCrel-1 overexpression William G. Honer for the SP18 hybridoma supernatant. We are causes dopamine-dependent neurotoxicity and suggest that in- grateful to Fritz Ochsenbein for preparing the figures. This work was hibition of dopamine secretion by CDCrel-1 may contribute to supported by grants from the Swiss National Science Foundation to the development of AR-JP. However, further studies will be H.B. and by the National Center for Competence in Research (NCCR) necessary to elucidate the detailed molecular mechanism(s) on Neural Plasticity and Repair.

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